Studies on the mode of action of tralkoxydim using cell suspension cultures of rice and flax

Cell suspension cultures of rice (Oryza sativa) and oil-flax (Linum usitatissimum) were established and grown in the dark. Sensitivity of these cultures to the cereal selective cyclohexanedione graminicide tralkoxydim (2-[1-(ethoxyimino) propyl]-3 hydroxy-5-mesitylcyclohex-2-enone) was investigated in different stages of the growth cycle by assessing culture fresh weight (FW), dry weight (DW) and settled cell volume (SCV). Both cultures of rice and flax were most tolerant in the early linear phase with an lo of ca. 100 muM, measured by settled cell volume (SCV). In other stages of the growth cycle rice and flax cultures were two to three times more sensitive; the I50 was 51 and 72 muM tralkoxydim in the middle and late linear phase for rice and ca. 30 muM in the lag, linear, and stationary phase for flax. Growth of flax was completely inhibited above 25 muM while rice showed growth even at 200 muM tralkoxydim. Individually, the growth parameters of FW and DW were more strongly inhibited compared to SCV in rice while the reverse situation was true for flax cultures with SCV strongly inhibited. The rice and flax cultures were inoculated with 14C-tralkoxydim, on days 2, 4 and 7 of the growth cycle and incubated for 6, 24 and 72 h periods. Following solvent extraction in hexane/dichloromefliane and or adsorption/elution of polar products on C18 columns, extracts of cells, cell debris and media were assayed for 14C-activity and subsequently separated and identified by thin layer chromatography (tic). The cultures of rice and flax showed a difference in distribution pattern of 14C-activity between three fractions: debris, cells and media. The cell debris of rice and flax contained ca. 10% of the recovered radioactivity. The rice cell and media fractions contained similar amounts of radioactivity (45%) but the flax media fraction contained ca. 75% and the cell fraction ca. 20% of recovered radioactivity. Recovery of radioactivity from the cultures was 50 and 80 % for rice and flax respectively. The difference could have been caused by a reduced number of solvent extraction steps for the flax medium but it is also possible that rice may have been biologically more active in transforming 14C-tralkoxydim. The concentration of radioactivity in the debris fractions decreased with culture age but remained constant in the cell fractions. Radioactivity accumulated in rice but decreased in flax cells with increasing incubation time (6-72 h). Both rice and flax cell suspension cultures produced at least 8 degradation or transformation products as assessed by tic, autoradiography and radiochromatogram scanning. The rice and flax cell suspension cultures produced the same metabolites. The limitations of the techniques used did not permit differential rates of metabolism to be distinguished within or between the tested species. CeU and media fractions of both species contained the same metabolites; a strong presence in the cells was often conversely accompanied by a weak presence in the media. The parent tralkoxydim could be transformed abiotically or biotically to yield oxazole and imine derivatives. Biotic transformation also produced hydroxy tralkoxydim and glutaric acid. In addition, cell fractions showed the progressive development of several polar compounds considered to be conjugation products. The differential response of rice and flax to growth inhibition in vitro could not readily be explained by the subtle qualitative differences in the transformation products identified nor by quantitative differences in 14C-labelled distribution between cell debris, cell and media extracts. This in vitro study provided a useful technique to investigate the transformation of tralkoxydim and the characterisation of its metabolites. The techniques applied herein could usefully evolve by expanding the species range in vitro and the introduction of chemical methods of manipulating metabolism e.g. via cytochrome P450 inhibitors and stimulators. Such in vitro studies could form valuable adjuncts to the classical in vivo studies investigating the mode of action of herbicides

Microbiome profiling by Illumina sequencing of combinatorial sequence-tagged PCR products

We developed a low-cost, high-throughput microbiome profiling method that uses combinatorial sequence tags attached to PCR primers that amplify the rRNA V6 region. Amplified PCR products are sequenced using an Illumina paired-end protocol to generate millions of overlapping reads. Combinatorial sequence tagging can be used to examine hundreds of samples with far fewer primers than is required when sequence tags are incorporated at only a single end. The number of reads generated permitted saturating or near-saturating analysis of samples of the vaginal microbiome. The large number of reads al- lowed an in-depth analysis of errors, and we found that PCR-induced errors composed the vast majority of non-organism derived species variants, an ob- servation that has significant implications for sequence clustering of similar high-throughput data. We show that the short reads are sufficient to assign organisms to the genus or species level in most cases. We suggest that this method will be useful for the deep sequencing of any short nucleotide region that is taxonomically informative; these include the V3, V5 regions of the bac- terial 16S rRNA genes and the eukaryotic V9 region that is gaining popularity for sampling protist diversity.Comment: 28 pages, 13 figure

The electronic structure of (C59N)2 from high energy spectroscopy

We report the results of a detailed study of the occupied and unoccupied electronic structure of dimers of the new heterofullerene C59N by means of photoemission and electron energy-loss spectroscopy. A close similarity is found between the electronic structures of pristine (C59N)2 and C60 with an additional broadening of the spectra in the former due to the distortion of the fullerene cage caused both by dimerization and the chemical substitution. Both the occupied and unoccupied electronic states, as well as the interband transitions between them, attest to the high degree of molecular character retained in the solid state. Comparison of the shake-up structures in the C1s and N1s X-ray photo emission spectra confirm that the highest lying occupied states in the heterofullerene have a strong degree of N character, whereas the lowest lying unoccupied states have mainly C character. We also present the optical conductivity of the heterofullerene (derived from the loss function), which shows an optical gap of 1.4 eV, some 0.4 eV smaller than that of C60